Gonadotropin releasing hormone (hereinafter called GnRH) participates in the hypothalamic-pituitary gonadal control of human reproduction. The involvement of GnRH has been demonstrated in several carcinomas and GnRH analogue treatment has been applied in breast, prostatic, pancreatic, endometrial and ovarian cancers (Kadar et al. Prostate 12:229-307, 1988). These analogues suppress tumor cell growth in vitro and in vivo. The existence of GnRH binding sites was revealed in the corresponding malignant cells and in well-established cell lines (Emons et al. J. Clin. Endocrinol. Metab. 77:1458-1464, 1993), though preliminary results suggest that the GnRH receptor involved may differ from the previously documented receptor (Kakar et al. Biochem. Biophys. Res. Comm. 189:289-295, 1992).
Although GnRH binding sites have been demonstrated in a number of solid tumors and various carcinoma cell lines derived mainly from hormone dependent tissues, their existence in colon or renal carcinoma has not been previously documented. The presence of specific GnRH binding sites in colon, breast, prostate, ovarian endometrium, renal and liver carcinomas, is shown here. Surprisingly, the specific GnRH binding sites are not limited to hormone-dependant tissues, as indicated by the marked killing of colon adenocarcinoma, renal cell adenocarcinoma and hepatocarcinoma cells.
W093/15751 describes various conjugates of GnRH, a linking group and Pseudomonas exotoxin A, prepared using the techniques of synthetic organic chemistry, used for the sterilization of animals by killing gonadotropin releasing cells of the animals pituitary gland.
The present invention describes the construction, by the techniques of genetic engineering, of PE based chimeric toxins, aimed at targeting those neoplastic cells bearing GnRH binding sites. The chimeric toxins of the present invention are fusion proteins and, as such, do not contain a chemical linking group (as in the above-mentioned patent). Therefore, they are completely different proteins from the molecules described in W093/15751.
Using different kinds of targeting moieties, a large number of immunotoxins have been generated in the last 20 years by chemical linkage techniques or recombinant DNA technology. The size of these targeting moieties varies widely, ranging from large antibodies to small growth factors, cytokines and antibody fragments.
The ability of large chimeric proteins, as the Met-GnRH-PE constructions described in the present invention, to target cells via a very small portion of the polypeptide (a peptide of ten amino acids, as used as the targeting moiety of the present invention), and yet retain their original functions, namely binding and internalization, open up new possibilities in designing targeted immunotoxins.
Colon, breast, and prostate cancer—three out of the four major malignancies occurring in humans, together with ovarian, endometrium, renal and liver carcinomas, account for more than 50% of cancer related death. The presence of specific GnRH binding sites in all of these cancers, may suggest a more general role of GnRH and/or GnRH-like peptides in the malignant process.
Collectively, these results disclose what could be considered the Achilles' heel of these malignant growths, a finding that could open up new vistas in the fight against cancer.
In view of their efficient growth inhibition of the above-mentioned cancer cells and their specificity regarding the non-target cells, the novel Met-GnRH-PE chimeric toxins are promising candidates for cancer treatment.